Peptide-Chelator conjugates

ABSTRACT

Peptide-chelator conjugates are provided that when labelled with a traceable metal are useful for diagnostic imaging of sites of inflammation. The peptide component is an antagonist of the naturally occurring tetrapeptide tuftsin while the chelator component serves as a labelling site for metals, in particular radionuclide metals such as technetium-99m.

FIELD OF THE INVENTION

This invention is in the field of diagnostic imaging, and relates to apeptide targetting agent useful for targetting sites of inflammation.

BACKGROUND OF THE INVENTION

The art of diagnostic imaging exploits targetting agents that in bindingor localizing sites selectively within the body, help to resolve theimage of diagnostic interest. Monoclonal antibodies for example havebeen developed to have high affinity and specificity for particularcancer cells and therefore are useful for imaging tumours. Despite highaffinity and specificity, antibodies do not provide ideal imaging agentssince they are costly to produce on a commercial scale as well as theirpoor labelling characteristics. In particular, metal labels tend to bindat numerous low-affinity binding sites on antibodies and are released invivo resulting in undesirable accumulation of the label at non-targetsites. An alternative targetting agent to antibodies are small receptorbinding peptides. Peptides offer the advantage of efficient labellingfacilitated by conjugation to various chelating molecules. Otheradvantages of peptides over antibodies is their ease of synthesis, rapidtissue penetration and rapid clearance from the body.

A naturally occurring tetrapeptide, tuftsin TKPR (Seq. ID no: 1), wasdiscovered to stimulate phagocytosis by binding to receptors expressedon the outer surface of neutrophils and macrophages. Phagocytosisconstitutes a major line of defense for a host against bacterialinfections, therefore as a stimulator of phagocytosis tuftsin would beexpected to be a good peptide for imaging sites of infectiousinflammation. However, studies show that tuftsin labelled with aradionuclide metal undesirably accumulates in non-target tissue. Inparticular, labelled tuftsin accumulated in the gastrointestinal tractwhich limits its usefulness as an imaging agent.

In light of the difficulties associated with antibodies, it would bedesirable to provide a peptidic targetting agent capable of localizingat sites of inflammation while not having substantial accumulation innon-target tissue.

SUMMARY OF THE INVENTION

Peptide-chelator conjugates are provided that when labelled with atraceable metal are useful for diagnostic imaging of sites ofinflammation. The peptide component is an antagonist of the naturallyoccurring tetrapeptide tuftsin while the chelator component serves as alabelling site for metals, in particular radionuclide metals such astechnetium-99m. According to an aspect of the invention, there areprovided peptidechelator conjugates in which Thr-Lys-Pro-Pro-Arg (Seq IDno: 2) is coupled at its N-terminus to a metal chelator.

In a particular embodiment of the present invention, the metal chelatorcomponent of 15 the conjugate is of the formula I: ##STR1## wherein R₁and R₂ together form a 5- or 6-membered heterocyclic ring which isoptionally fused to a 5- or 6-membered ring wherein either ring isoptionally substituted with groups selected from alkyl, alkoxy,carboxyl, halogen, hydroxyl and a linking group;

R₃ is selected from H; alkyl; and alkyl substituted by a group selectedfrom amino, aminoacyl, carboxyl, guanidinyl, hydroxyl, thiol, phenyl,phenolyl, indolyl and imidazolyl;

R₄ is selected from hydroxyl, alkoxy, and a linking group; and

T represents H or a sulfur protecting group.

In a particular embodiment of the present invention, the metal chelatorcomponent of the conjugate is of the formula II: ##STR2## wherein R₃, R₄and T are as defined above; and

According to an aspect of the invention, the peptide-chelator conjugatesare provided in combination with a diagnostically useful metal or anoxide or nitride thereof.

According to an aspect of the present invention, there is provided amethod of imaging a site of inflammation in a mammal, comprising thestep of administering a diagnostically effective amount of a compositioncomprising a peptide-chelator conjugate in which a peptide of theformula Thr-Lys-Pro-Pro-Arg (Seq ID no: 2) is coupled at its N-terminusto a metal chelator which is complexed to a diagnostically useful metalor an oxide or nitride thereof.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides peptide-chelator conjugates that when complexedwith a diagnostically useful metal are useful for imaging sites ofinflammation. The peptide-chelator conjugate, also referred to as"conjugate", incorporates an antagonist of tuftsin coupled at itsN-terminus to any metal chelator, the peptide component consisting ofthe amino acid sequence Thr-Lys-Pro-Pro-Arg represented by the singleletter coed TKPPR (Seq ID no: 2) hereinafter referred to as "thepeptide". It is understood that the peptide may be extended at itsC-terminus by 1 to 3 amino acid residues or may be modified at theC-terminus for example amidated such that the targetting activity of thepeptide is not substantially inhibited. In an embodiment of the presentinvention, the peptide is coupled to the metal chelators (I) illustratedabove which is disclosed in co-pending United States application in thename of Pollak et al, filed on 22 December 1993, incorporated herein byreference and metal chelators (II).

The terms defining the variables R₁ -R₄ and T as used hereinabove informula (I) and (II) have the following meanings:

"alkyl" refers to a straight or branched C₁ -C₈ chain and includes lowerC₁ -C₄ alkyl;

"alkoxy" refers to straight or branched C₁ -C₈ alkoxy and includes lowerC₁ -C₄ alkoxy;

"thiol" refers to a sulfhydryl group that may be substituted with analkyl group to form a thioether;

"sulfur protecting group" refers to a chemical group that is bonded to asulfur atom and inhibits oxidation of sulfur and includes groups thatare cleaved upon chelation of the metal. Suitable sulfur protectinggroups include known alkyl, aryl, acyl, alkanoyl, aryloyl, mercaptoacyland organothio groups.

"linking group" refers to a chemical group that serves to couple thepeptide to the chelator while not adversely affecting either thetargetting function of the peptide or the metal binding function of thechelator. Suitable linking groups include alkyl chains and amino acidchains functionalized with a reactive groups for coupling to the peptideor chelator.

"metal chelator" refers to a molecule that forms a stable complex with atraceable metal atom under physiological conditions in that the metalremains bound to the conjugate in vivo.

In preferred embodiments of the invention, the chelators conform to theabove formulae (I) and (II) in which: R₁ and R₂ together form asix-membered heterocyclic ring; R₃ is selected from H and a hydroxylsubstituted alkyl group selected from methyl and ethyl and mostpreferably hydroxymethyl; R₄ is a linking group of one to three aminoacid residues and T is the sulfur protecting group acetamidomethyl (Acm)or benzoyl (Bz);

In more preferred embodiments of the invention, the chelators conform tothe above formula (I) wherein R₁ and R₂ together form a pyridine ring;R₃ is hydroxymethyl; T is Acm and R₄ is a linking group selected from-Gly- and -Gly-Asp-Gly- (Seq ID no: 3). These chelators in a formcoupled to the peptide may be represented by the sequences:

    Pic-Ser-Cys(Acm)-Gly-TKPPR                                 (Seq ID no: 4)

and

    Pic-Ser-Cys(Acm)-Gly-Asp-Gly-TKPPR                         (Seq ID no: 5)

wherein Pic represents the amino acid derivative picolinic acid.

In a preferred embodiment of the invention, the chelators conform to theabove formula (II) wherein R₃ is hydroxymethyl; T is Acm or Bz and R₄ isthe linking group -Ser-Gly-Asp-Gly- (Seq ID no: 6). This chelatorcoupled to the peptide at the linking group of R₄ is represented by thesequence:

    Bz-MA-Ser-Cys-Ser-Gly-Asp-Gly-TKPPR                        (Seq ID no: 7)

wherein Bz-MA represents the group benzoylmercaptoacetic acid.

Peptide-chelator conjugates of the invention may be prepared by variousmethods depending upon the chelator chosen. The peptide portion of theconjugate is most conveniently prepared by techniques generallyestablished in the art of peptide synthesis, such as the solid-phaseapproach. Solid-phase synthesis involves the stepwise addition of aminoacid residues to a growing peptide chain that is linked to an insolublesupport or matrix, such as polystyrene. The C-terminus residue of thepeptide is first anchored to a commercially available support with itsamino group protected with an N-protecting agent such as at-butyloxycarbonyl group (tBoc) or a fluorenylmethoxycarbonyl (FMOC)group. The amino protecting group is removed with suitable deprotectingagents such as TFA in the case of tBOC or piperidine for FMOC and thenext amino acid residue (in N-protected form) is added with a couplingagent such as dicyclocarbodiimide (DCC). Upon formation of a peptidebond, the reagents are washed from the support. After addition of thefinal residue, the peptide is cleaved from the support with a suitablereagent such as trifluoroacetic acid (TFA) or hydrogen fluoride (HF).

Peptide and chelator components are coupled to form a conjugate byreacting the free amino group of the Thr residue of the peptide with anappropriate functional group of the chelator such as a carboxyl group oractivated ester. For example, a conjugate may incorporate the chelatorethylenediaminetetraacetic acid (EDTA), common in the art ofcoordination chemistry, when functionalized with a carboxyl substituenton the ethylene chain. Synthesis of EDTA derivatives of this type arereported in Arya et al, (Bioconjugate Chemistry 1991, 2:323) wherein thefour coordinating carboxyl groups are each blocked with a t-butyl groupwhile the carboxyl substituent on the ethylene chain is free to reactwith the amino group of the peptide thereby forming a conjugate.

A conjugate may incorporate a metal chelator component that is peptidicie. compatible with solid-phase peptide synthesis. In this case thechelator may be coupled to the peptide in the same manner as EDTAdescribed above or more conveniently the chelator and peptide aresynthesized in toto starting from the C-terminal residue of the peptideand ending with the N-terminal residue of the chelator.

Conjugates may further incorporate a linking group component that servesto couple the peptide to the chelator while not adversely affectingeither the targetting function of the peptide or the metal bindingfunction of the chelator. Suitable linking groups include amino acidchains and alkyl chains functionalized with reactive groups for couplingto both the peptide and the chelator. An amino acid chain is thepreferred linking group when the chelator is peptidic so that theconjugate can be synthesized in toto by solid-phase techniques.

An alkyl chain linking group may be incorporated in the conjugate byreacting the amino group of the Thr residue of the peptide with a firstfunctional group on the alkyl chain such as a carboxyl group or anactivated ester. Subsequently the chelator is attached to the alkylchain to complete the formation of the conjugate by reacting a secondfunctional group on the alkyl chain with an appropriate group on thechelator. The second functional group on the alkyl chain is selectedfrom substituents that are reactive with a functional group on thechelator while not being reactive with the Thr residue of the peptide.For example, when the chelator incorporates a functional group such as acarboxyl group or an activated ester, the second functional group of thealkyl chain linking group can be an amino group. It will be appreciatedthat formation of the conjugate may require protection and deprotectionof the functional groups present in order to avoid formation ofundesired products. Protection and deprotection is accomplished usingprotecting groups, reagents and protocols common in the art of organicsynthesis. Particularly, protection and deprotection techniques employedin solid phase peptide synthesis described above may be used.

An alternative chemical linking group to an alkyl chain is polyethyleneglycol (PEG) which is functionalized in the same manner as the alkylchain described above for incorporation in the conjugates. It will beappreciated that linking groups may alternatively be coupled first tothe chelator and then to the peptide.

In accordance with one aspect of the invention, peptide-chelatorconjugates incorporate a diagnostically useful metal capable of forminga complex. Suitable metals include radionuclides such as technetium andrhenium in their various forms such as ^(99m) TcO³⁺, ^(99m) TcO₂ ⁺,ReO³⁺ and ReO₂ ⁺. Incorporation of the metal within th conjugate can beachieved by various methods common in the art of coordination chemistry.When the metal is technetium-99m, the following general procedure may beused to form a technetium complex. A peptide-chelator conjugate solutionis formed initially by dissolving the conjugate in aqueous alcohol suchas ethanol. The solution is then degassed to remove oxygen then thiolprotecting groups are removed with a suitable reagent, for example withsodium hydroxide and then neutralized with an organic acid such asacetic acid (pH 6.0-6.5). In the labelling step, a stoichiometric excessof sodium pertechnetate, obtained from a molybdenum generator, is addedto a solution of the conjugate with an amount of a reducing agent suchas stannous chloride sufficient to reduce technetium and heated. Thelabelled conjugate may be separated from contaminants ^(99m) TcO₄ ⁻ andcolloidal ^(99m) TcO₂ chromatographically, for example with a C-18 SepPak cartridge.

In an alternative method, labelling can be accomplished by atranschelation reaction. The technetium source is a solution oftechnetium complexed with labile ligands facilitating ligand exchangewith the selected chelator. Suitable ligands for transchelation includetartarate, citrate and heptagluconate. In this instance the preferredreducing reagent is sodium dithionite. It will be appreciated that theconjugate may be labelled using the techniques described above, oralternatively the chelator itself may be labelled and subsequentlycoupled to the peptide to form the conjugate; a process referred to asthe "prelabelled ligand" method.

Another approach for labelling conjugates of the present inventioninvolves techniques described in a co-pending United States applicationSer. No. 08/152,680 filed 16 November 1993, incorporated herein byreference. Briefly, the peptide-chelator conjugates are immobilized on asolid-phase support through a linkage that is cleaved upon metalchelation. This is achieved when the chelator is coupled to a functionalgroup of the support by one of the complexing atoms. Preferably, acomplexing sulfur atom is coupled to the support which is functionalizedwith a sulfur protecting group such as maleimide.

When labelled with a diagnostically useful metal, peptide-chelatorconjugates of the present invention can be used to detect sites ofinflammation by procedures established in the art of diagnostic imaging.A conjugate labelled with a radionuclide metal such as technetium-99mmay be administered to a mammal by intravenous injection in apharmaceutically acceptable solution such as isotonic saline. The amountof labelled conjugate appropriate for administration is dependent uponthe distribution profile of the chosen conjugate in the sense that arapidly cleared conjugate may be administered in higher doses than onethat clears less rapidly. Unit doses acceptable for imaging inflammationare in the range of about 5-40 mCi for a 70 kg individual. In vivodistribution and localization is tracked by standard scintigraphictechniques at an appropriate time subsequent to administration;typically between 30 minutes and 180 minutes depending upon the rate ofaccumulation at the target site with respect to the rate of clearance atnon-target tissue.

The following examples are presented to illustrate certain embodimentsof the present invention.

    ______________________________________                                        EXAMPLE 1                                                                     Preparation of Conjugates:                                                    ______________________________________                                        Pic--Ser--Cys(Acm)--G--TKPPR                                                                         (Seq ID no: 4)                                         Pic--Ser--Cys(Acm)--GDG--TKPPR                                                                       (Seq ID no: 5)                                         Bz--MA--Ser--Cys--SGDG--TKPPR                                                                        (Seq ID no: 7)                                         ______________________________________                                    

Peptide-chelator conjugates were synthesized in toto using9-fluorenylmethyloxycarbonyl (FMOC) chemistry on an2-methoxy-4-alkoxybenzyl alcohol resin preloaded with the protectedC-terminus residue (Sasrin resin, Bachem Biosciences Inc., PhiladelphiaPa.) using an Applied Biosystems 433A peptide synthesizer (Foster City,Calif.). N-terminus residues Pic and Bz-MA were incorporated by usingpicolinic acid and benzoylmercaptoacetic acid respectively as the finalresidue in the synthesis.

The resin bound conjugate was dried in vacuo for 12 hours. Cleavage ofthe conjugate from the resin involved mixing a cooled solution of 95%trifluoroacetic acid (TFA) and 5% water (1 ml per 100 mg ofpeptide-resin) with the conjugate-resin for 1.5 to 2 hours at roomtemperature. The resin was removed by filtration and washed 3 times with30 ml t-butyl methyl ether in a 50 ml conical polypropylene centrifugetube forming a white precipitate. The precipitate was dissolved in waterwith added acetonitrile. The precipitate was frozen in acetone-dry iceand lyophilized over 12 hours. The resulting white powder was dissolvedin water, filtered through a 0.45 μm syringe filter (Gelman Acrodisc LCPVDF) and purified by reversed-phase HPLC (Beckman System Gold) with aC₁₈ column (Waters RCM 25×10) using 0.1% TFA in water as buffer A and0.1% TFA in acetonitrile as buffer B. The column was equilibrated with100:0 buffer A: buffer B and eluted with a linear gradient in 25 min at1 ml/min to 50% buffer B. Fractions were reanalysed on the HPLC andpooled according to matching profiles. When necessary the pooledfractions were repurified using the same conditions. The pure fractionswere frozen in acetone-dry ice and lyophilized over 10 hours to give awhite powder.

EXAMPLE 2 Labelling and Imaging of Conjugates

Imaging studies were performed in a rat inflammation model as follows.Male Wistar rats (Charles River, 150-200g) were injected intramuscularlywith a virulent E. coli (ATCC 25922, 0.1 ml of 0.5×10⁹ organisms/ml)suspension into their left hindlegs 24 hours before imaging. Focalinflammation in the leg was visually detectable after 1 day.

Each conjugate (50 μl, 2 mg/ml saline) was placed in a 1.5 ml tube with100 μl saline, 100 μl pertechnetate (10 mCi) and 100 μl stannousgluconate (50 μg stannous chloride and 1 mg sodium gluconate). The tubewas capped and placed in a boiling water bath for 10 minutes and thenfiltered through a Watman PVDF syringe to collect the labelled conjugatesolution which was further diluted with saline to prepare an injectablesolution (200 μl) containing about 100 μCi (3.7 MBq) of activity. Therats were anaesthetized with somnotol (40 to 50 mg/kg), and the Tc-99mlabelled conjugate solution (200 μL) was injected intravenously via thetail vein. Serial whole-body scintigrams were acquired 30 minutes afteradministration with a gamma camera. The rats were then killed withanaesthesia and samples of organs, urine, blood, inflamed muscle (leftleg) and non-inflamed muscle (right leg) were weighed and counted ineither a well-type gamma counter or in a gamma dose calibrator. Theblood dose calculations were made based on assumptions, (1) that the ratweighed 200 g and (2) that the blood volume constituted 8% of bodyweight. Results presented in the following table are averages ofmultiple trials and are corrected for the residual dose in the tail.

    __________________________________________________________________________                                                    % dose/g                                                                          In-                                                                           flam                                                           Time    G.I.   mus-                                                                             Uninfl                                                                            Inflam:            Chelator    Linking Group   Peptide  (min)                                                                             Urine                                                                             tract                                                                            Blood                                                                             cle                                                                              muscle                                                                            Uninfl             __________________________________________________________________________    PicSerCys(Acm)                                                                            Gly             TKPR.sup.e.                                                                            35  22.8                                                                              22.8                                                                             0.268                                                                             0.120                                                                            0.040                                                                             3.0                PicSerCys(Acm)                                                                            Gly             KPPR.sup.b.                                                                            45  43.5                                                                              -- 0.220                                                                             0.125                                                                            0.059                                                                             2.1                PicSerCys(Acm)                                                                            Gly             TKPRTKPR.sup.c.                                                                        35  16.1                                                                              4.2                                                                              0.136                                                                             0.071                                                                            0.022                                                                             3.2                PicSerCys(Acm)                                                                             ##STR3##       forTKPPR 35  12.2                                                                              11.3                                                                             0.748                                                                             0.213                                                                            0.086                                                                             2.5                PicSerCys(Acm)                                                                             ##STR4##       forTKPPR 35  14.3                                                                              9.2                                                                              0.485                                                                             0.163                                                                            0.062                                                                             2.6                PicSerCys(Acm)                                                                            Gly             RTKPR.sup.d.                                                                           35  45.4                                                                              15.0                                                                             0.169                                                                             0.099                                                                            0.033                                                                             3.0                BzMASerCys  SGDG.sup.g      TKPPR.sup.f                                                                            35  47.8                                                                              -- 0.483                                                                             0.226                                                                            0.057                                                                             4.0                PicSerCys(Acm)                                                                            GDG             TKPPR.sup.f                                                                            35  63.1                                                                              -- 0.288                                                                             0.166                                                                            0.034                                                                             4.9                PicSerCys(Acm)                                                                            Gly             TKPPR.sup.f                                                                            30  44.71                                                                             5.08                                                                             0.392                                                                             0.154                                                                            0.052                                                                             3.6                PicSerCys(Acm)                                                                            Gly             TKPPR.sup.f                                                                            180 80.62                                                                             3.71                                                                             0.048                                                                             0.026                                                                            0.006                                                                             4.7                __________________________________________________________________________     .sup.a. Seq ID no: 8                                                          .sup.b. Seq ID no: 9                                                          .sup.c. Seq ID no: 10                                                         .sup.d. Seq ID no: 11                                                         .sup.e. Seq ID no: 1                                                          .sup.f. Seq ID no: 2                                                          .sup.g. Seq ID no: 6                                                     

Results provided in the table reveal that conjugates in which the TKPPR(SEQ ID No: 2) peptide is coupled N-terminally to a chelator exhibitedsignificantly higher ratios of inflamed to uninflamed muscle readings(target to background) as well as superior distribution profilescompared to conjugates incorporating other peptides. Because of thesesuperior properties, the TKPPR (SEQ ID No: 2)-containing conjugates gaveimages that most clearly differentiated the inflamed muscle tissue.These desirable imaging results were obtained regardless of whether theTKPPR peptide (SEQ ID No: 2) was coupled to an N₂ S₂ or an N₃ S classchelator; both exhibited high target to background and rapid clearanceinto the urine. The presence and nature of the linking group also didnot adversely affect the imaging results with TKPPR (SEQ ID No: 2);conjugates incorporating different linking groups, varying from 1 to 4amino acids long, also yielded good images.

On the other hand, conjugates containing a metal chelator coupled to thenative tuftsin peptide or to peptides structurally related to TKPPR (SEQID No: 2) showed target to background ratios significantly lower thanthe TKPPR (SEQ ID No: 2) conjugates, and showed high accumulation in theGI tract. The one exception, the TKPRTKPR (SEQ ID No: 10) (tuftsindimer) conjugate, did exhibit low accumulation in the GI tract andblood, nevertheless the particularly low urine levels suggest undesiredaccumulation at an unknown location. When coupled to the chelator at itsC-terminus, the peptide TKPPR (Seq ID no: 2) also exhibited the lessdesirable properties of relatively low target to background and high GItract accumulation, resulting in a less resolved image.

The results indicate that conjugates incorporating the peptide TKPPR(Seq ID no: 2), coupled to a chelator at its N-terminus, are useful forimaging inflammation and that this indication is independent of the typeof chelator or linking group incorporated in the conjugate.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 11                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ThrLysProArg                                                                  (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ThrLysProProArg                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GlyAspGly                                                                     1                                                                             (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note="Ser substituted with                            picolinic acid (Pic)."                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note="Cys substituted with                            acetamidomethyl (Acm)."                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       SerCysGlyThrLysProProArg                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note="Ser substituted with                            picolinic acid (Pic)."                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note="Cys substituted with                            acetamidomethyl (Acm)."                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       SerCysGlyAspGlyThrLysProProArg                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       SerGlyAspGly                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note="Ser is substituted with                         benzoylmercaptoacetic acid (Bz-MA)."                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note="Cys is substituted with                         acetamidomethyl (Acm)."                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       SerCysSerGlyAspGlyThrLysProProArg                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note="Lys is optionally                               substituted at its epsilon amino group with                                   Gly-Gly-."                                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GlyLysAla                                                                     1                                                                             (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       LysProProArg                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      ThrLysProArgThrLysProArg                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      ArgThrLysProArg                                                               15                                                                            __________________________________________________________________________

We claim:
 1. A peptide-metal chelator conjugate useful for imaging ofsites of inflammation, wherein said metal chelator is coupled to theN-terminus of a peptide consisting of the sequence Thr-Lys-Pro-Pro-Arg(SEQ ID No: 2), wherein the metal chelator has the general formula:##STR5## wherein R₃ is selected from H; alkyl; and alkyl substituted bya group selected from amine, aminoacyl, carboxyl, guanidinyl, hydroxyl,thiol, phenyl, phenolyl, indolyl and imidazolyl;R₄ is selected fromhydroxyl, alkoxy and a linking group; and T represents H or a sulfurprotecting group.
 2. A peptide-metal chelator conjugate according toclaim 1, wherein the peptide is coupled to the metal chelator at R₄. 3.A peptide-metal chelator conjugate according to claim 1, wherein thepeptide is coupled to the metal chelator by a linking group at R₄.
 4. Apeptide-metal chelator conjugate according to claim 3, wherein thelinking group is one or more amino acid residues.
 5. A peptide-metalchelator conjugate according to claim 3, wherein the linking group isselected from -Gly-, -Gly-Asp-Gly- (SEQ ID No: 3) and -Ser-Gly-Asp-Gly-(SEQ ID No: 6).
 6. A peptide-metal chelator conjugate complex, whereinsaid peptide-metal chelator conjugate is useful for imaging of sites ofinflammation, and wherein said metal chelator is coupled to theN-terminus of a peptide consisting of the sequence THr-Lys-Pro-Pro-Arg(SEQ ID NO: 2), and wherein the metal chelator has the metal chelatorhas the general formula: ##STR6## wherein R₃ is selected from H; alkyl;and alkyl substituted by a group selected from amino, aminoacyl,carboxyl, guanidinyl, hydroxyl, thiol, phenyl, phenolyl, indolyl andimidazolyl;R₄ is selected from hydroxyl, alkoxy and a linking group; andT represents H or a sulfur protecting group; and a diagnostically usefulmetal or an oxide or nitride thereof.
 7. A peptide-metal chelatorconjugate useful for imaging of sites of inflammation, having theformula:

    Bz-MA-Ser-Cys-SGDG-TKPPR (SEQ ID NO: 2).


8. 8. A peptide-chelator conjugate according to claim 7, in a formcomplexed with a diagnostically useful metal or an oxide or nitridethereof.